1. Field of the Invention
The invention relates to laboratory apparatus used in the measurement of transport of substances across membranes, for example, membranes of biological tissues, or a thin section or sheet of a synthetic material, and tissue or cell cultures on a support therefor.
2. Description of Related Art
Hobson, U.S. Pat. No. 4,667,504 issued May 26, 1987 discloses an apparatus for determining in vitro the penetration rate of chemicals across a biological membrane. The apparatus comprises two housings, one holding a reservoir of test chemical, and the other providing a chamber for flowing receptor solution across a membrane held in a membrane holding compartment. The membrane holding compartment comprises a cylindrical depression surrounding an open end of the receptor solution chamber. The receptor solution chamber is tilted slightly with its higher end open to the membrane holding compartment. The tile prevents bubbles in the receptor solution from becoming stalled or strapped inside the chamber and interfering with the reliability and reproducibility of tests. An inlet bore from the upper surface of the receptor housing leads to a closed end of the chamber near the membrane holding compartment to the upper surface of the receptor housing. The inlet bore is sized smaller than the outlet bore. The depth of the depression forming the membrane holding compartment varies, tapering from a lesser depth at its intersection with the chamber to a greater depth at the outer circumference of the depression. The thus formed truncated cone ensures that a sample biological membrane is stretched taut over the chamber opening by the force fastening the reservoir housing to the receptor solution housing.
The publication of Ussing and Zerahn (Acta Physiol. Scand. 23:110-127(1951) describes an apparatus for the determination of sodium flux and short circuit current in frog skin. The device has an area for placement of the skin between two opposing half cells, pressed against the skin by two lucite screws held by steel uprights. The tips of the lucite screws are conical and fit into conical depressions in the center of the ebonite dishes. Buffer solutions are circulated and aerated by air entering through side tubes. Bridges, held in position by pieces of rubber tubing, fit tightly into short celluloid side tubes sealed into the two chambers and these connect to a reservoir system. The device is also fitted with a series of electrodes.
Schoenwald and Huang (J. Pharm. Sci., 72:11 (1983)) discloses a device for mounting of corneas to determine transport of material across corneal tissue. The system is composed of two acrylic plastic blocks. Each block acts as an opposing half cell with and area for the corneal tissue between. The cornea is mounted using a system of three rings, and the ring system with the cornea is positioned in the opening of the blocks and form a watertight seal when sufficient lateral pressure is applied to the blocks in a manner similar to that described above by Ussing and Zehran. Fluid reservoirs on each side of the tissue are mixed with a gas lift mechanism, also similar to Ussing and Zehran above. Stirring motors mounted on the blocks, connect by shafts with blades on the terminus, through the block to the reservoir at the tissue face. These blades promote mixing at the tissue surface. The reservoirs are heated by circulation of temperature controlled fluid through channels in the block. The fluid reservoirs and fluid circulation channels for temperature control are within the same contiguous block.
Hildago et. al., (Gastroenterology 96:736-49 (1989)), disclose a method for the measurement of transport of material across a monolayer formed by cultured cells. Cells are grown in an appropriate media in a plastic cylinder which is caped at one end with a filter membrane. When the cells reach a confluent monolayer, the cells and the cylinder with attached filter are placed in another larger diameter cylinder with fluid which acts as the receiver solution. Materials placed in the smaller cylinder, with the cells are transported across the cells and the filter membrane to the reservoir solution of the larger cylinder.
Grass and Sweetana (Pharm. Res., 5:6 (1988) disclose a diffusion cell (chamber) for the measurement of tissue permeability. The apparatus is comprised of at least one chamber with a first and a second volume element, each of which contains a reservoir for fluids and a means to circulate fluids. Also included is a means to retain a membrane separating the first and second volume elements whereby the fluid contained in the reservoir in the first volume element housing could communicate with the fluid contained the reservoir in the second volume element in the absence of the membrane. Also included are a membrane separating the first and second volume elements, a means to circulate fluid contained in each of the first and second volume elements, and a means to attach the first and second volume elements on a contiguous surface of each of the first and second volume elements in an adjacent facing relationship, wherein the first and second volume element housings are separated from each other at their contiguous surfaces by the membrane.
The disclosure of the above publications is incorporated herein by reference
Measurement of the transport properties of molecules through various barriers is often conducted in an in vitro experiment using a device such as one of those described above. Devices which expedite the experimental process are advantageous. Such devices can relate to the measurement of transport properties through biological tissue, cell cultures, or synthetic membranes. Most commonly, the use of a side-by-side type of diffusion apparatus or cell is used. By the expression "side-by-side" type of diffusion apparatus (or cell) is meant an apparatus having two housing elements separated by the tissue or membrane to be tested.
For biological tissues, it has been possible to use the device and method of Ussing and Zehran described above. This method has certain disadvantages. The method of Ussing and Zehran uses a device which is usually comprised of several different materials all of which are in contact with the circulating fluids. Generally it is accepted that contact with as few materials as possible is preferred. Additionally, the design of the Ussing system and similarly derived designs, incorporate a temperature control means as an integral part of the fluid reservoirs, but this temperature control means does not directly encompass the tissue containing sections of the device, where the experiments actually take place. Thus, the environment of the chamber on which the experiment takes place is prone to fluctuate with respect to temperature. Furthermore, the Ussing chamber requires that the connecting tubing be disconnected, or that cleaning be done individually within the reservoir units.
Also, the Ussing device (apparatus) and those other devices derived from the Ussing device frequently allow the entrapment of bubbles in the device, with elimination of these bubbles sometimes being quite difficult. These bubbles can affect the experiment, especially if they are adjacent to the tissue surface. This is most frequent, although not limited to horizontally mounted tissues.
Another problem associated with known devices is a more technical problem relating to mixing and fluid flow within the chambers. Fluid mixing is generally recognized to be critical to the outcome of many of these types of experiments. In such known devices, mixing is accomplished by a variety of mechanisms, including stirring rod, or bars, or gas lift as described above. Such previous designs have created turbulent flow patterns, or those which directly impact on the tissue causing potentially accelerated damage.
Thus there is a need for an improved device (or apparatus) for use in in vitro experiments to test for transport of materials across tissues and synthetic membranes and cultured cells.